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Objectives To compare and evaluate phenotypic and genotypic methods for the detection of antimicrobial resistance (AMR) in Campylobacter jejuni and Campylobacter coli in England and Wales. Methods WGS data from 528 isolates of Campylobacter spp. (452 C. jejuni and 76 C. coli) from human (494), food (21) and environmental (2) sources, collected between January 2015 and December 2016, and from the PHE culture collection (11) were mapped to genes known to be associated with phenotypic resistance to antimicrobials in the genus. Phenotypic antibiotic susceptibility (erythromycin, ciprofloxacin, tetracycline, gentamicin and streptomycin) testing using an in-agar dilution method was performed on all isolates. Results Concordance between phenotypic resistance and the presence of corresponding AMR determinants was 97.5% (515/528 isolates). Only 13 out of 528 isolates (10 C. jejuni and 3 C. coli) had discordant interpretations for at least one of the five antibiotics tested, equating to a total of 15 (0.6%) discrepancies out of 2640 isolate/antimicrobial combinations. Seven discrepant results were genotypically resistant but phenotypically susceptible (major errors) and eight discrepant results were genotypically susceptible but phenotypically resistant (very major errors). Conclusions The use of this bioinformatics approach for predicting AMR from WGS data for routine public health surveillance is a reliable method for real-time monitoring of changing AMR patterns in isolates of C. jejuni and C. coli.

Antibiotic resistance in aquatic bacteria has increased steadily as a consequence of the widespread use of antibiotics, but practice and international treaty should have limited antibiotic contamination in Antarctica. We estimated antibiotic resistance in microorganisms isolated from the Antarctic marine waters and a penguin rookery, for 2 reasons: (i) as a measure of human impact and (ii) as a potential “snapshot” of the preantibiotic world. Samples were taken at 4 established sampling sites near Palmer Station, which is situated at the southern end of the Palmer Archipelago (64°10′S, 61°50′W). Sites were chosen to provide different potentials for human contamination. Forty 50 mL samples of seawater were collected and colony-forming units (CFU)/mL were determined at 6 and 20 °C. For this study, presumed psychrophiles (growth at 6 °C) were assumed to be native to Antarctic waters, whereas presumed mesophiles (growth at 20 °C but not at 6 °C) were taken to represent introduced organisms. The 20–6 °C CFU/mL ratio was used as a measure of the relative impact to the ecosystem of presumably introduced organisms. This ratio was highest at the site nearest to Palmer Station and decreased with distance from it, suggesting that human presence has impacted the natural microbial flora of the site. The frequency of resistance to 5 common antibiotics was determined in each group of isolates. Overall drug resistance was higher among the presumed mesophiles than the presumed psychrophiles and increased with proximity to Palmer Station, with the presumed mesophiles showing higher frequencies of single and multiple drug resistance than the psychrophile population. The frequency of multidrug resistance followed the same pattern. It appears that multidrug resistance is low among native Antarctic bacteria but is increased by human habitation.

Seasonal shifts in rhizosphere microbial populations were investigated to follow the influence of plant developmental stage. A field study of indigenous microbial rhizosphere communities was undertaken on pea (Pisum satvium var. quincy), wheat (Triticum aestivum var. pena wawa) and sugar beet (Beta vulgaris var. amythyst). Rhizosphere community diversity and substrate utilization patterns were followed throughout a growing season, by culturing, rRNA gene density gradient gel electrophoresis and BIOLOG™. Culturable bacterial and fungal rhizosphere community densities were stable in pea and wheat rhizospheres, with dynamic shifts observed in the sugar beet rhizosphere. Successional shifts in bacterial and fungal diversity as plants mature demonstrated that different plants select and define their own functional rhizosphere communities. Assessment of metabolic activity and resource utilization by bacterial community-level physiological profiling demonstrated greater similarities between different plant species rhizosphere communities at the same than at different developmental stages. Marked temporal shifts in diversity and relative activity were observed in rhizosphere bacterial communities with developmental stage for all plant species studied. Shifts in the diversity of fungal and bacterial communities were more pronounced in maturing pea and sugar beet plants. This detailed study demonstrates that plant species select for specialized microbial communities that change in response to plant growth and plant inputs.

Aquatic habitats are important potential sites for gene transfer between indigenous bacteria and released genetically engineered microorganisms (GEMs). Legislation governing GEM release, and other practical considerations, have resulted in microcosms, of varying complexity, being used to study gene transfer in aquatic environments. This article reviews these microcosms, with particular emphasis on the more complex designs and, where possible, compares gene transfer results obtained in them with in situ studies. We conclude that microcosms can give results that are consistent with those obtained in situ and thus can be relied upon to give realistic predictions of in situ behaviour.

Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 × 10 7 g −1 ), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 × 10 8 g −1 , which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.

There is evidence for alteration of bacterial genomes through evolutionary times and some of the pressures and molecular mechanisms underlying these changes have been investigated. Models such as the Barlow-and-Hall system are proving very useful in understanding evolution and predicting change in natural populations of bacteria. Use of genomics, study of the fossil record, identification of environmental stresses, past, present, and future, and modeling are all important to increasing our knowledge and understanding of the mechanisms and consequences of bacterial genome diversification. Using the most modern molecular techniques to define bacteria taxa, the eubacteria are divided into 23 phyla. For the past 50 years nomenclature of bacteria has been in constant flux as new species and genera are added or removed for the standard systematics. These changes reflect the latest fashions in classification and the application of new molecular and physiological methods for characterizing bacteria. Insight into the richness of genetic potential in the world around us is essential if we are to protect our natural environmental heritage and leave to our posterity a world that is vital and healthy.

Gene inactivation and deletion are just as important to genome evolution as are duplication and other mutational events. The environment and phenotype (delivered by accessory gene sequences) act selectively in concert to enable E. coli to diversify both ecologically and genetically. Having strategies for acquiring genetic information of all sorts of sizes is helpful to cells in rapidly adapting to new environmental niches. If sequences provide selective advantage to the cell then the genes become established and persist. When a cell has ceased to grow in what is considered a normal way, in nature or in laboratory experiments, it then is posed as a serious physiological problem. The types of species present describe the ecological state of the biofilm; for example, anaerobes to the bottom and aerobes to the top. A biofilm is an organized structure, and quorum sensing is involved. As in many microbiological investigations, the advent of easy sequencing, the applications of bioinformatic analysis, and array technology are going to make a huge contribution to one's understanding of microbial interactions. Group activities are effectively promoted by interactions between different species and genera.

For a bacterium short-term survival depends on the high fidelity of the replication machinery to prevent genomic alteration, but in the long term, evolution needs to occur. There are two basic themes, one providing for genome modification and the other providing for an increase in genomic content. The processes considered in this chapter are those of replication, amplification, and deletion. In fact, if gene acquisition, through transfer processes, is as high as is considered likely, then processes driving deletion reactions will be strategically important to conserving genome size. DNA sequences are subject to change for better or for worse, so selection would appear to favor individuals with a mutation rate approaching zero and an accurate replication machinery. Mutation is an event that is more likely to adversely effect the activity of a gene product. As mutations occur there is no mechanism for identifying them or replacing them by recombination. Clonal species are strongly driven by competition because one clone is "much like" another. This allows them to acquire by recombination large amounts of genetic sequence. Homology-independent (site-specific) pathways of DNA incorporation are likely to lead to increases in genome complexity. But nonhomologous (site-specific) recombination is also important in shaping bacterial genomes. Evidence suggests that gene elongation and duplication was the route taken by ancestral life forms to enlarge their genomes and increase their biochemical capacity. Genomic analysis of the three cell domains, Archaea, Bacteria, and Eukarya is allowing an analysis of their relationships.

Microbial ecologists and geneticists were forced to look at the natural environment to determine experimentally whether horizontal gene exchange could take place in nature. Horizontal gene transfer can be confirmed in many environments. The data to date suggest that various mechanisms of horizontal gene transfer with various outcomes and consequences can take place in all natural environments, whether aquatic, terrestrial, or biological, often in biofilms. Transformation has been shown to occur in freshwater and marine environments and in various soils. The presence of free DNA in many environments suggests that the potential for transformation is widespread. It is likely that this chapter discovers that not only is horizontal gene transfer more widespread than we now can document but that the themes and variations in its mechanisms are much more robust than we can now imagine. In any case it is now clear that horizontal gene transfer has been and continues to be a strong and important force in the evolution of bacterial genomes. The concepts of the importance of transduction and transformation have greatly changed during the past few years. The discovery that competence is often controlled by quorum-sensing mechanisms has greatly advanced our understanding of the underlying molecular mechanisms of transformation and excited many microbiologists to search for new species with new mechanisms and signals for the initiation of competence and, hence, genetic transformation. Such insights have excited interest in understanding how bacteria and other microorganisms exchange genetic information in nature.

Self-transmissible plasmids conferring mercury resistance were exogenously isolated from the bacterial populations of sugar beet roots (rhizoplane) and leaves (phyllosphere) into a Pseudomonas putida recipient. Fifty rhizoplane plasmids and 29 phyllosphere plasmids (60–383 kb) were purified. Numerical analysis of plasmid DNA restriction enzyme digest patterns identified five distinct groups. Three of these plasmid groups were isolated from sugar beet crops grown at the same site over three consecutive years, demonstrating their established presence. Each group of plasmids comprised individual isolates with structural additions or deletions. The frequency of exogenous isolation correlated with factors likely to influence plant growth, bacterial activity and the physiological state of donors prior to sampling. All plasmids investigated conferred narrow spectrum mercury resistance with a reductase detoxification mechanism. None of the plasmids conferred resistance to a range of antibiotics, other heavy metals, or to UV, and following transfer to recipient bacteria the range of carbon source utilisation was not altered. This is the first report of the persistence of Pseudomonas spp. plasmid structural types isolated over several years from a terrestrial habitat.

The kinetics of conjugation, retrotransfer and mobilisation were studied at 5–15 min intervals between strains of Pseudomonas putida using plasmid pQKH6, isolated from river epilithon, and R300B. Transconjugants from the direct conjugation of pQKH6 and mobilisation of R300B by pQKH6 appeared rapidly, reaching maximum densities within 30–60 min of the start of both filter and liquid mating experiments. However, retrotransconjugants only appeared after a delay. This delay was short (approx. 45–60 min) in filter mating and much longer (2–5 h) for liquid mating experiments. Attempts at predicting the time course of retrotransconjugant development from (1) numbers of transconjugants from the conjugation and mobilisation experiments and (ii) mathematical models based on a mass action approach, both failed to reproduce the observed delay. It was concluded that retrotransfer did not proceed by either a one-step mechanism occuring early in conjugation or two separate conjugation and mobilisation steps. The clear demonstration of a delay in retrotransconjugant formation implies that a new mechanism must be sought. The likely importance of retrotransfer in the environment is discussed.

1 Introduction 2 Bacteriocins 3 Bacteriophages 4 Conclusions Keywords: bacteriocins and bacteriophages; bacteriocins-proteins to kill or inhibit competing strains; membrane potential destruction; protein synthesis inhibition; bacteriophages (phages)-viruses in bacterial cells and biosynthetic systems for reproduction; single-stranded RNA phages; phage role in natural bacterial populations; bacteriocins and bacteriophages-affecting growth and biochemical relationships

Antibiotic resistance in aquatic bacteria has increased steadily as a consequence of the widespread use of antibiotics, but practice and international treaty should have limited antibiotic contamination in Antarctica. We estimated antibiotic resistance in microorganisms isolated from the Antarctic marine waters and a penguin rookery, for 2 reasons: (i) as a measure of human impact and (ii) as a potential "snapshot" of the preantibiotic world. Samples were taken at 4 established sampling sites near Palmer Station, which is situated at the southern end of the Palmer Archipelago (64 degrees 10'S, 61 degrees 50'W). Sites were chosen to provide different potentials for human contamination. Forty 50 mL samples of seawater were collected and colony-forming units (CFU)/mL were determined at 6 and 20 degrees C. For this study, presumed psychrophiles (growth at 6 degrees C) were assumed to be native to Antarctic waters, whereas presumed mesophiles (growth at 20 degrees C but not at 6 degrees C) were taken to represent introduced organisms. The 20-6 degrees C CFU/mL ratio was used as a measure of the relative impact to the ecosystem of presumably introduced organisms. This ratio was highest at the site nearest to Palmer Station and decreased with distance from it, suggesting that human presence has impacted the natural microbial flora of the site. The frequency of resistance to 5 common antibiotics was determined in each group of isolates. Overall drug resistance was higher among the presumed mesophiles than the presumed psychrophiles and increased with proximity to Palmer Station, with the presumed mesophiles showing higher frequencies of single and multiple drug resistance than the psychrophile population. The frequency of multidrug resistance followed the same pattern. It appears that multidrug resistance is low among native Antarctic bacteria but is increased by human habitation.

Biofilm formation on presterile slate-discs placed at a pristine source site and at three polluted sites located upstream from, at, and down-stream from a sewage-works outfall in a South Wales river, was monitored over 14 days. Viable bacterial cell densities in biofilms increased with time at all sites but more rapidly at the polluted sites, and stabilized after 1-4 days at levels equal to those of stones indigenous to the corresponding sites, and similar to those seen in other comparable pristine and polluted rivers. Biofilm cell densities were elevated about 1,000-fold at the sewage works outfall compared with the site immediately upstream. Downstream from the outfall, the cell densities were intermediate between the upstream and outfall values. Epilithon resuspended from the slates during colonization was tested for its capacity to biodegrade the surfactant sodium dodecyl sulphate (SDS) in die-away tests. No biodegradation of this common pollutant was observed for the samples from the pristine source. In contrast, even after only 1 day, all polluted sites produced samples capable of biodegrading SDS. Longer exposure in the river led to more rapid onset of biodegradation in the die-away tests, indicating an adaptation mechanism(s). Die-away kinetics were fitted by computerized non-linear regression analysis to one of several models. The model of best fit involved biodegradation of SDS by a bacterial population growing at the expense of endogenous carbon. The regression parameter reflecting SDS-degrading activity of the epilithic samples increased markedly during Days 0-4 for all three polluted sites. The stabilized values (Days 4-14) increased from the upstream site to the outfall, then decreased to intermediate values downstream. Although this pattern corresponded to the changes in viable cell numbers, the effect of the sewage input was less marked for the SDS-degrading activities than for bacterial cell densities. In addition, there was little variation in growth characteristics throughout colonization at all three polluted sites. Collectively the results indicate that the observed adaptation during exposure in the river is attributable to colonization of the epilithon by an existing SDS-degrading population, rather than the acquisition or adaptation of this biodegradative capability.

SUMMARY: A nonlinear technique to calculate parameters for segregational instability and differences in cellular growth-rate for plasmid-bearing micro-organisms growing in batch or continuous culture is presented. This method is compared with an approximate technique based upon linear regression. The accuracy and sensitivity of the results are evaluated by use of simulated data and biological data taken from experiments with Pseudomonas aeruginosa(pGSS15) and Escherichia coli(pHSG415). It is demonstrated that the nonlinear analysis gives results which are significantly more accurate and which show much better agreement with the data. Consequently, the new analysis leads to quite different conclusions with regard to the nature of the instability of the plasmid-bearing strain. This method offers an opportunity to study the genetic and physiological aspects of plasmid instability and so aid the design and optimization of cloning vectors.

To provide estimates of horizontal gene transfer from transgenic crops to indigenous soil bacteria, transformation frequencies were obtained for naturally transformable Acinetobacter baylyi BD413 using a chromosomally integrated plant transgene. The transgene comprised sequences for two phenotypic markers: kanamycin resistance (npt II) and green fluorescent protein (gfp ), expressed from their own bacterial promoters. Recipient bacteria carried a copy of these two genes, with deletions in their 3'-termini abolishing the marker activity, these genes were integrated into a 16S rRNA gene in the bacterial chromosomal genome or carried on a broad host range plasmid. Successful recombination between the plant transgene and the bacterial genome resulted in restoration of the markers, allowing detection through antibiotic selection and fluorescence. Transformation parameters of increasing complexity, without any enrichment steps, were used to approach the field conditions, while still obtaining measurable transformation frequencies. In pure culture filter experiments, transformation was detected using ground, chopped and whole leaves, as well as whole sterile seedlings, and ground roots. In sterile soil microcosms, transformation was detected using pure plant DNA (3.6 × 10-8 transformants per recipient) and ground leaves (2.5 × 10-11 ). Transformation was also detected for the first time in non-sterile soil using pure plant DNA (5.5 × 10-11 ). Since the same constructs were used throughout, these data allow predictions of even more complex environmental systems where measurable frequencies are not easily obtainable.

In this study we present a bacteriophage isolated from the Great Salt Plains National Wildlife Refuge (GSP) that is shown to have a genome size of 340 kb, unusually large for a bacterial virus. Transmission electron microscopy analysis of the virion showed this to be a Myoviridae, the first reported to infect the genus Halomonas. This temperate phage, PhigspC, exhibits a broad host range, displaying the ability to infect two different Halomonas spp. also isolated from the GSP. The phage infection process demonstrates a high level of tolerance towards temperature, pH and salinity; however, free virions are rapidly inactivated in water unless supplemented with salt. We show that susceptibility to osmotic shock is correlated with the density of the packaged DNA (rho(pack)). Lysogens of Halomonas salina GSP21 were detrimental to host fitness at 10% salinity, but the lysogen was able to grow faster than the wild type at 20% salinity. From these results we propose that the extensive genome of PhigspC may encode environmentally relevant genes (ERGs); genes that are perhaps not essential for the phage life cycle but increase host and phage fitness in some environmental conditions.

Objectives: A little-understood mode of antimicrobial resistance in Staphylococcus aureus is the evolution of a sub-population of small-colony variants (SCVs). SCVs are a cause of persistent and recurring infections refractory to antimicrobial chemotherapy. Following the inadvertent isolation of suspected SCVs growing in the presence of triclosan we set out to evaluate the formation of these colonial mutants and assess their antimicrobial susceptibility. Methods: SCVs were isolated on Mueller–Hinton agar supplemented with 1 mg/L triclosan. SCV formation frequency was calculated using a selection of both clinical methicillin-resistant S. aureus (MRSA) isolates and methicillin-susceptible S. aureus strains. Antimicrobial susceptibility was assessed and the fabI gene of SCVs was sequenced to ensure resistance was not mediated by mutation of this gene. Results: We have found in vitro that triclosan can select for S. aureus colonies showing the characteristic SCV phenotype with low-level triclosan resistance and which coincidently have reduced susceptibility to penicillin and gentamicin. Additionally, triclosan-isolated SCVs were shown to have an increased tolerance to the lethal effects of triclosan. Conclusions: We propose the formation of SCVs by S. aureus is a novel mechanism of resistance to low concentrations of triclosan, which for 25 years has been used widely in the domestic setting in various consumer healthcare products. More recently it has been recommended for the control of MRSA. Consequently, our results identify the potential for treatment failure in infections already notoriously difficult to eradicate. It remains unclear to what extent isolates with decreased susceptibility to triclosan would develop and have the fitness to survive under real world conditions.

Nine mercury-resistance plasmids isolated from river epilithon were assessed for their ability to retrotransfer the non-conjugative IncQ plasmid, R300B, derivatives of which have commercial uses that may result in accidental or deliberate release into the environment. Retrotransfer frequencies ranging from 2.1×10−4 to 1.75×10−5 were obtained for five of the nine plasmids – the remaining plasmids showed low or undetectable retrotransfer ability. The majority of the retrotransfer-proficient plasmids could not be classified by the tests used. Classical incompatibility testing with RP4 identified pQKH6, pQKH54 and pQM719 as IncP-1. Hybridization to replicon probes confirmed this for pQKH6 and pQM719 and added pQKH33. PCR with primers designed to amplify trfA and korA regions of IncP-1 plasmids did not identify any other plasmids. Plasmids pQKH6 and pQM719 but not pQKH54 produced similar SphI restriction profiles to the IncP-1β subgroup. The complete nucleotide sequence of pQKH54 was determined, revealing it to have a complete IncP-1 backbone but belonging to a new distinct subgroup which was designated IncP-1γ. The results emphasize the ubiquity and diversity of IncP-1 plasmids in the environment but demonstrate that plasmids of as yet unknown groups are also able to retrotransfer IncQ plasmids efficiently.

Publisher Summary This chapter discusses the transformation in aquatic environments. Transformation is one of three mechanisms that promote gene exchange in bacterial populations, performed by many Gram-positive and Gram-negative species. It is an active, highly evolved, and dedicated process of gene exchange, governed by chromosomal genes. “Free” DNA, released from either a living or a lysed “dead” cell, is taken up only by a competent recipient cell. The environment is “awash” with eukaryotic and prokaryotic DNA thus, mechanisms to identify the DNA as “self” or “nonself” have evolved. In some species, this “free” DNA has an internal sequence that allows it to be selectively identified and taken up. Competence is often a transient and tightly regulated stage through which a proportion of a population passes because of physiological changes occurring within those individual cells.

We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium. Further, we provide evidence of in situ competition between these phages. This is the first in situ demonstration of such microbial interactions in soil. To achieve this, sugar beet seeds were inoculated with Serratia liquefaciens CP6RS or its lysogen, CP6RS-ly-Φ1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages. Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. A Podoviridae phage, identified as ΦCP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, the Siphoviridae phage ΦCP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some ΦCP6-1 phage up to day 137, yet hardly any ΦCP6-4 phages were found, and this was consistent with previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model.

Bayston et al.describe the isolation of SCVs with low-level resistance to tri-closan, reduced coagulase production, reduced haemolyticactivity and did not detect auxotrophy. They recognize, as othershave done, the possible clinical importance of SCVs and thepotential role of triclosan in their generation. However, wewould like to urge some caution in their interpretation of theirdata and consequently on the significance of the issues raised.A significant difference between triclosan and other antimi-crobials known to select for SCVs (principally aminoglycosides)is that it is used topically. It is thought that SCVs are unlikely toinitiate infections, rather, once a wild-type infection has beenestablished, antibiotic treatment selects for mutation to give theSCV phenotype/form. Hence, selection of SCVs by systemicantibiotics occurs once the bacteria have broached the skin andare at the site of infection, whereas triclosan-induced SCVs willpresumably be on the skin surface. Thus, an important questionis whether these SCVs would then be capable of ever initiatingan infection in healthy individuals.Extending this logic means the triclosan-induced SCVsformed at the skin surface may be transmissible by skin–skincontact, as wild-type S. aureus. This could provide a mechanismfor them coming into contact with wounds or finding otherroutes through the epidermis. However, it would also leave themvulnerable to the current mechanisms of preventing transfer ofinfection, such as alcohol hand-washes and barrier systems suchas gloves.The association of SCVs with triclosan-impregnated polymersis possibly more worrying than selection of SCVs by topicalpreparations. For example, the use of triclosan-impregnatedsutures

The aquatic environment dominates the Earth, both in terms of its global proportion and also the population sizes and diversity of life maintained within it. Bacteriophage are viruses that prey on bacterial hosts and are widespread in natural systems; the aquatic environment being no exception [4, 6]. There are a wide variety of morphological phage types [1] and many of those that look similar can be demonstrated to have taxonomically different host species. The natural relationships between phage and their hosts has received little attention despite their abundance and widespread distribution. For example in the northern Adriatic Sea, electron microscopy-studies have shown that natural populations of bacteria appear to have a significant degree of phage infection. The percentage of rods with mature phages visible internally was significantly correlated to increasing rod densities [42]. Up to 27% of rods, 79% of cocci and 100% of spirillae were visibly infected with viruses. The highest overall infection frequency of the entire bacterial community was 30%. This calculation assumed that mature phage are only visible during the last 14 to 27% of the latent period [29].

SUMMARY: The effect of dilution rate on the instability kinetics of Escherichia coli RV308(pHSG415) during glucose-limited continuous culture is examined. Two nonlinear models are fitted to the data, both of which characterize the plasmid-host system in terms of the rate parameters R (for the plasmid segregation rate) and dμ (for the specific growth rate difference between plasmid-free and plasmid-bearing single cells). In the first model, both R and dμ have constant values with respect to time. In the second, either R or dμ is represented as a time-dependent function. Although both models fit the data equally well, it is demonstrated that the constant rate parameter model gives results which appear to be misleading. A comparison is also made among some of the many plasmid instability models (both mass-balance and segregated) which have appeared in the literature. It is found that all of these give identical trajectories and differ only in the definitions of the rate parameters used.

The capacities of epilithic and planktonic river bacterial populations to degrade sodium dodecyl sulfate (SDS) in samples taken at two times during 1987 from one clean and four polluted sites in a South Wales river were estimated in die-away tests under simulated environmental conditions. There was a relatively slow disappearance of SDS in die-away tests for both planktonic and epilithic populations taken from the clean source site, as compared with those taken from the downstream polluted sites, for which the rate of biodegradation was accelerated, sometimes after an apparent initial lag period. The kinetic components contributing to the die-away curves were quantified by nonlinear regression analysis in which the experimental data were fitted to a variety of possible kinetic models. All samples except for one from the polluted sites best fitted a model which describes the biodegradation of SDS at concentrations well below its K m by bacteria whose growth is exponential and unaffected by the addition of a test substrate. Each sample from the clean source site fitted a different model, but there was generally little or no growth on endogenous carbon. A consideration of the numerical values of constants derived from the modeling of epilithic and planktonic populations from polluted sites indicated clearly that the biodegradative capacity of epilithic bacterial populations towards SDS is more stable than that of planktonic bacterial populations.

This chapter is aimed at emphasising some of the principle features of the forgoing chapters that we consider most important and to put them into a general context. We do not intend to imply that the subjects that we discuss here are the only important issues in this book, only that they are the topics that interest us most.

Section I: General Aspects of Studying Genetics in Nature.- 1 Genetic approaches to the study of gene transfer in microbial communities.- 2 Factors influencing the dissemination of DNA by bacterial conjugation.- 3 Factors limiting gene transfer in bacteria.- 4 Phage genetics and ecology.- Section II: Aquatic Habitats.- 5 Plasmid transfer in the epilithon.- 6 Laboratory standardised biofilms as a tool to investigate genetic transfers in water systems.- 7 Survival of laboratory and freshwater bacteria carrying an extrachromosomal xylE gene in freshwater microcosms.- 8 Gene transfer in marine environments.- 9 Gene transfer in activated sludge.- Section III: Terrestial Habitats.- 10 Plasmid transfer between soil bacteria.- 11 Gene transfer in polluted soils.- 12 The potential for gene exchange between rhizosphere bacteria.- 13 The use of a Sesbania rostrata microcosm for studying gene transfer among microorganisms.- 14 Plasmid transfer to indigenous bacteria in soil and rhizosphere: problems and perspectives.- 15 Use of wide host range promoters to monitor the fate of recombinant DNA in soil.- 16 The role of soil bacteria in risk assessment analysis.- 17 Gene transfer between streptomycetes in soil.- 18 The survival of genetically engineered microorganisms and bacteria on inanimate surfaces and in animals.- Section IV: Conclusions.- 19 Plasmid transfer and the release of genetically engineered bacteria in nature: a discussion and summary.

Plasmids have been commonly found in bacteria from a variety of natural habitats. In estuarine water and sediment from Chesapeake Bay 46% of heterotrophs contained plasmids,1 whilst in a South Wales river sediment only 10–15% possessed these replicons2. It is probable that many of these plasmids are conjugative as Burton et al.2 found 86% were larger than 30 kb. Furthermore, Jobling et al3 found that 25% of the mercury resistant bacteria from the River Mersey transferred plasmid encoded, mercury resistance to Escherichia coli. It is also likely that most Gram negative genera are potential recipients for at least some of these naturally occurring plasmids. Support for this comes from a recent study in which the broad host range, self transmissible plasmid R68 was found to transfer from Pseudomonas aeruginosa into 38% of the freshly isolated, heterotrophic bacteria tested.4 The genera which accepted the plasmid were Pseudomonas, Acinetobacter, Alcaligenes, Chromobacter- ium, Achromobacter and Moraxella, which are all very common aquatic bacteria.5

The temporal and geographical distributions of planktonic bacteria, and their ability to degrade sodium dodecyl sulphate (SDS) (AS(+) phenotype), in a polluted South Wales river, is reported for five sites source to estuary sampled during a one-year period. The annual mean prevalence of AS(+) isolates at all sites was 8.1-16.0% of the total number of isolates, and these values were not altered by including SDS in culture media. Although the proportion of AS(+) isolates in clean water at the source was not significantly different to that of polluted sites, the AS(+) cell density was lower at the source because of its lower overall numbers. The percentage of AS(+) isolates in estuarine water was higher than at the three polluted mid-river sites, but when cell numbers were taken into account, the AS(+) cell density was the same at all polluted sites including the estuary. There was no correlation between percentage of AS(+) and either BOD or oxygen concentration, but AS(+) isolates were significantly more prevalent at the end of summer. Of the AS(+) isolates, more than half contained constitute alkysulphate enzymes, the remainder being induced or repressed by SDS; the relative proportions of enzyme regulatory type did not vary significant between sites or at different sampling times.

A microcosm using rotating slate discs in a chemostat was used to study bacterial population dynamics and genetic interactions in river epilithon. Populations of all introduced donor and recipient Pseudomonas spp. decreased with time but all the bacteria survived better on the slate discs than in the liquid phase. Conjugal transfer of an epilithic plasmid encoding mercury resistance (pQM1) occured with transfer frequencies of 1.4 × 10−6 to 3.6 × 10−3 per recipient, which were about 100-fold lower than in standard membrane filter mating experiments.

A survey of riverine bacteria showed that the ability to grow in the presence of the surfactant sodium dodecyl sulphate (SDS) was widespread but the proportion of SDS-resistant isolates was significantly greater at a polluted site, as compared with clean source water. Bacteria containing the alkylsulphatases that initiate SDS degradation were also widespread. The polluted site yielded significantly more alkylsulphatase-containing strains than the clean site, and this difference was due to a greater number of strains at the polluted site bearing constitute rather than inducible enzymes. In addition, the incidence of strains containing multiple alkylsulphatase was much higher at the polluted than at the clean site.

A naturally occurring mercury-resistance, conjugative plasmid, designated pQM1, was isolated from a bacterial population on the surface of stones from a river using Pseudomonas aeruginosa as a recipient. This was a narrow-host-range plasmid [IncP-13; 165 MDa; Tra+, Hgr, fluorescein mercuric acetater, merbrominr, Phi(E79), UVr] confined to some Pseudomonas spp. It was used to demonstrate transfer between bacteria on stones in laboratory microcosm experiments and in situ. Transfer occurred (3.3 X 10(-1) to 6.8 X 10(-9) per recipient) at all the temperatures used (6-20 degrees C), although frequencies were lower in the cold. Nutrient status also affected transfer frequency, rich conditions promoting transfer. The presence of competing bacteria in the natural epilithon lowered transfer frequencies, but when unscrubbed stones were heat treated, transfer was enhanced, perhaps because of nutrient release from the heated epilithon.

Epilithic bacteria were isolated nonselectively from riverbed stones and examined by gel zymography for their ability to produce alkylsulfatase (AS) enzymes and thus to metabolize alkyl sulfate surfactants such as sodium dodecyl sulfate. The percentages of AS+ isolates from stone epilithon at five sites from the source to the river mouth were measured on five sampling days spread over 1 year. The results showed that (i) the prevalence of epilithic AS+ strains (as a percentage of all isolates) was much higher at polluted sites than at the source; (ii) when averaged over the whole river, percentages of AS+ strains were significantly higher at the end of summer compared with either the preceding or the following winter; (iii) analysis of site-sampling time interactions indicated that water quality factors (e.g., biochemical oxygen demand and dissolved oxygen concentration) rather than climatic factors determined the distributions of epilithic AS+ isolates; (iv) constitutive strains were the most prevalent (7.2% of all isolates), with smaller numbers of isolates with inducible (4.5%) and repressible (1.7%) enzymes.